The radical scavenging activity of monocaffeoylquinic acids: the role of neighboring hydroxyl groups and pH levels

Caffeoylquinic acids (CQAs) are well-known antioxidants. However, a key aspect of their radical scavenging activity – the mechanism of action – has not been addressed in detail thus far. Here we report on a computational study of the mechanism of activity of CQAs in scavenging hydroperoxyl radicals. In water at physiological pH, the CQAs demonstrated ≈ 104 times higher HOO˙ antiradical activity than in lipid medium (k(lipid) ≈ 104 M−1 s−1). The activity in the aqueous solution was determined by the hydrogen transfer mechanism of the adjacent hydroxyl group (O6′–H) of the dianion states (Γ = 93.2–95.2%), while the single electron transfer reaction of these species contributed 4.8–6.8% to the total rate constants. The kinetics estimated by the calculations are consistent with experimental findings in water (pH = 7.5), yielding a kcalculated/kexperimental = 2.4, reinforcing the reliability and precision of the computational method and demonstrating its utility for evaluating radical reactions in silico. The results also revealed the pH dependence of the HOO˙ scavenging activity of the CQAs; activity was comparable for all compounds below pH 3, however at higher pH values 5CQA reacted with the HOO˙ with lower activity than 3CQA or 4CQA. It was also found that CQAs are less active than Trolox below pH 4.7, however over pH 5.0 they showed higher activity than the reference. The CQAs had the best HOO˙ antiradical activity at pH values between 5.0 and 8.6. Therefore, in the physiological environment, the hydroperoxyl antiradical capacity of CQAs exhibits similarity to renowned natural antioxidants including resveratrol, ascorbic acid, and Trolox.


Computational details
The thermochemical characteristics (bond dissociation energies (BDEs), ionization energies (IEs), and proton affinities (PAs)) of the compound were investigated at the M06-2X/6-311++G(d,p) level of theory.Additionally, the kinetic parameters of the compound, including their activation energies (DG s ) in kcal mol −1 , tunneling corrections (k), and rate constants (k), were computed.The activities of the compounds were modelled in the gas phase, the physiological environment (the lipid medium consisted of pentyl ethanoate).0][41][42] The kinetic calculations were conducted following the established technique for the quantum mechanics-based assay designed to evaluate the overall free radical scavenging activity (QM-ORSA) with the solvation model based on the density (SMD) method for pentyl ethanoate and water solvents.The aforementioned test has been widely employed in order to assess the antiradical properties of antioxidants. 34,35,39,40,43he rate constant (k) was determined through the application of the usual transition state theory (TST) under the conditions of a 1 M standard state, [44][45][46][47][48] and the details are shown in Table S1, ESI.† Here DG s represents the Gibbs free energy of activation, h denotes the Planck constant, k B represents the Boltzmann constant, s is the reaction symmetry number, 49,50 and k signies the tunneling corrections that were determined by the utilization of the Eckart barrier calculation method,. 51he computations were performed utilizing the Gaussian 16 suite of programs 52 and the Eyringpy code, depending on the particular circumstance. 53,54Atom-in-molecule (AIM) analysis 55 was performed by using the AIM2000 soware. 56

The thermodynamic study
Based on the core structure of CQA, the hexagon rings, HO, and COOH groups can undergo rotation to yield a variety of conformers.The most likely conformer to participate in a radical scavenging reaction is the most stable one, and thus electron energy levels of all possible conformers of each CQA were evaluated in the rst stage. 57Subsequently, the ve conformers with the lowest electronic energy were subjected to free energy analysis using the M06-2X/6-311++G (d,p) level of theory.Details are shown in the ESI (Fig. S2).† It was found that the DG°value of 3CQA (i.e. the structure as drawn in Fig. S2, ESI †) was determined to be the lowest among all the 3CQA conformers (3CQA-1-4) by 2.8-5.1 kcal mol −1 .Similarly, the lowest energy 4CQA and 5CQA conformers are drawn in Fig. S2, ESI.† The estimation of conformer relative populations using the Maxwell-Boltzmann distribution 58,59 revealed that the conformers 3CQA, 4CQA, and 5CQA dominate the populations (>95%) under standard conditions; consequently, these conformers were used in subsequent investigations.
The lowest BDE values were observed at the O6 0 (7 0 )-H bonds in all of the studied acids with BDE(O6 0 (7 0 )-H) = 79.1-81.9kcal mol −1 for the lipid medium and BDE(O6 0 (7 0 )-H) = 82.7-85.4kcal mol −1 for the aqueous solution (Fig. 2a1-c1).The active site can be attributed in all cases to the formation of intramolecular hydrogen bonds between the hydrogen atoms of the adjacent hydroxyl groups and the O6 0 (O7 0 ) radicals. 60,62hile the values for other O-H bonds ranged from 104.9 to 131.6 kcal mol −1 , the BDE(C-H) values were between 88.3 and 102.6 kcal mol −1 .Surprisingly the H-abstraction of the COO-H bond was less likely with the BDE(COO-H) = 112.3-114.4kcal mol −1 (to emphasize, this refers to hydrogen abstraction; proton dissociation is much more likely, see below).The BDE values in the water were slightly higher than those in the pentyl ethanoate solvent.As expected the PA and IE values in the polar medium were lower than those of the nonpolar environment.The deprotonation was in the order of COO-H > O7 0 -H > O6 0 -H in all of the studied compounds, whereas the IE values varied from 93.5 to 139.6 kcal mol −1 .
The evaluation in the Gibbs free energies (DG°, Fig. 2a2-c2) of the HOOc + CQAs reactions following either of the three pathways revealed that the HOOc radical trapping activity of CQAs is only spontaneous via the hydrogen transfer of the O6 0 (7 0 )-H bonds (DG°= −3.6 to −6.2 kcal mol −1 ), whereas the other FHT reactions cannot happen in the studied media due to the positive DG°values.The SET mechanism is not spontaneous either in any of the studied environments, thus this reaction of the neutral states of CQAs can be safely ignored in the kinetic study.It is important to notice that the PL reactions are not spontaneous either in any of the studied environments; however, the PA values were substantially lower than the corresponding BDE values, thus the deprotonation of CQAs should be considered in the aqueous solution.Previous studies indicated that the addition reaction into the a,b-unsaturated bond had no contributions to the ROOc radical (i.e., HOOc and CH 3 OOc) scavenging activity, particularly in the physiological environments, 19,24,63 and RAF reaction is not supported for the p system of aromatic rings. 64,65Thus, this reaction was omitted in our study.Hence, in the lipid medium, the H-abstraction of the O6 0 (7 0 )-H bonds should be used to compute rate constants, whereas, in the aqueous solution, proton dissociation should be assessed before the kinetic investigation.

The kinetics of antioxidant activity
3.2.1.The deprotonation of CQA.The dissociated form of acidic species frequently overshadows the antiradical activity of the neutral species in aqueous environments. 37,41Thus the protonation states of CQAs in water at the physiological pH were analyzed.The structure of CQAs permits protonation at the COOH (pK a1 ), O7 0 -H (pK a2 ), and O6 0 -H (pK a3 ) bonds (Fig. 2); the pK a1 values of CQAs were obtained from a previous study, 66 while the pK a2 and pK a3 values were computed according to the previous study. 30The data are displayed in Fig. 3 and Table 1.
The pK a1 values for 3CQA and 5CQA are 3.95, while those for 4CQA are 4.14 (Table 1).The range of pK a2 values is 7.97 to 8.22, while the range of pK a3 values is 12.27 to 12.59.The calculated pK a values of 5CQA (pK a2 = 8.22 and pK a3 = 12.27) closely align with the experimental results (pK a2 = 8.21 and pK a3 = 12.5), 67 providing evidence for the accuracy and validity of the computational approach.The mole fractions f(H 2 A − ) and f(HA 2− ) range between 0.788 and 0.868 and between 0.131 and 0.212, respectively, while the H 3 A and A 3− phases are not present in water at pH = 7.40.Therefore, the CQAs exist in both anionic and dianionic states in water with a pH of 7.4.These two states were examined in the subsequent investigation.
3.2.2.The kinetics of the reaction of CQAs with HOOc radical in the physiological environments.The kinetics of the reactions between CQAs and HOOc in the aqueous solution were investigated for all states, using the methodology employed in earlier research on phenolic compounds.The competitive FHT reaction was utilized to evaluate the kinetics for neutral states, while the SET reaction was employed for anion states. 25,31,35Using eqn (2) and ( 3), the total rate constants of the states (k total ) were determined, whereas eqn (4) was used to derive the rate constant containing the molar fraction (k f ).Fig. 4 depicts the optimized transition structures (TS), data are in Table 2.
Lipid environment: Water at physiological pH: As shown in Table 2, the calculations suggest that CQAs can be potent HOOc scavengers in the nonpolar environment, with k total = 1.09 × 10 4 -1.93 × 10 4 M −1 s −1 .The rate constant for the reaction between 5CQA and HOOc was found to be the highest, whereas the reaction between 3CQA and HOOc exhibited the lowest rate constant.Based on the calculated data, the HOOc radical trapping ability of CQAs in the lipid medium can be ranked as follows: 5CQA > 4CQA > 3CQA.Thus, the activity of CQAs in the nonpolar medium is comparable to reference antioxidants including ascorbic acid (k = 5.71 × 10 3 M −1 s −1 ), 40 resveratrol (k = 1.31 × 10 4 M −1 s −1 ), 64 and Trolox (k = 3.40 × 10 3 M −1 s −1 ). 68n water at pH = 7.40, the FHT mechanism of the neighboring hydroxyl group (O6 0 -H) of the dianion states determined the HOOc radical scavenging activity of the CQAs (G = 93.2-95.2%),while the SET reaction of these species contributed approximately 4.8-6.8% to the k total .It should be noted that the tunneling corrections (k) had a negligible impact on the Habstraction rate constant of the dianion state, the substantial imaginary frequencies (n > 3000 cm −1 ) of these transition states notwithstanding.This suggests that the remarkably swi reaction rates are caused solely by the excessively low Gibb activation energy (DG s = 1.0-5.4kcal mol −1 ) (k TST z k D , where k D denotes the diffusion rate).The HOOc radical scavenging activity of the CQAs was not inuenced by the monoanion states, despite the fact these states exist about 13.1% to 21.2% (Table 1) of the CQAs in water at pH = 7.4.All compounds exhibit outstanding HOOc antiradical activity with k total z 10 8 M −1 s −1 .4CQA had the maximum activity with k total = 5.32 × 10 8 M −1 s −1 that is approximately 4.19 and 1.05 times faster than 5CQA and 3CQA, respectively.In water at pH = 7.4, the radical trapping activity of CQAs against HOOc is ranked as follows: 4CQA > 5CQA > 3CQA.In water at physiological pH, the CQAs demonstrated z 10 4 times greater HOOc radical scavenging ability than in the nonpolar environment.In water, CQAs have greater HOOc antiradical capacity than Trolox (k = 8.96 × 10 4 M −1 s −1 ), 68 resveratrol (k = 5.62 × 10 7 M −1 s −1 ), 64 and ascorbic acid (k = 9.97 × 10 7 M −1 s −1 ), 40 but the fairly similar   ). 63Consequently, CQAs are promising natural antioxidants.

The effect of pH values on the reactions of CQAs with HOOc in water
The impact of solution pH on the rate constants was also evaluated.Eqn ( 5)-( 8) were employed in the computation of several key parameters, namely the rate constant (k), the rate constant specic to each protonation state (k state ), the total rate constant (k total ), and the overall rate constant (k overall ).The outcomes are displayed in Fig. 6 and Table 3.
The log(k total ) for the total rate constant (Fig. 6a) did not change below pH 4.7, however, it increased signicantly between pH = 4.8 and 8.3 by 4-6 units and aerward grew progressively until pH 14.The sudden increase in the log(k total ) gures at pH = 4.8 and 8.3 is due to the appearance of HA 2− states and thus the onset of rapid SET processes.In acidic media (pH < 4.7), the reactions between CQAs and HOOc are sluggish because the majority of the CQAs exist in the H 3 A states (neutral states), acting via a slow FHT reaction.
It was demonstrated that pK a (HOOc) is 4.80, and thus the f(HOOc) value is zero at pH > 9.1.Since only pH levels below 9.1 had any impact on the k overall values of reactions between CQAs and HOOc, only these were looked at (Fig. 6b).It was found that as the pH levels rose, the k overall changed.Most of the studied acids showed a rise in log(k overall ) at pH 4; aer a brief fall, the log(k overall ) signicantly rose at pH = 4.7-6.5, before decreasing  Paper RSC Advances once again.For this range, k overall was 0 because f(HOOc) = 0 at pH > 9.2 (Fig. 6b).
In terms of the CQAs, the exhibition was fairly similar to the HOOc antiradical activity at pH < 3, however at the rest pH values, 5CQA reacted with the HOOc lower than 3CQA or 4CQA.It is important to notice that the 3CQA acid had a fairly similar HOOc radical scavenging activity to 4CQA in all of the studied pH levels.Compared with typical antioxidant-Trolox, at pH 4.7, CQAs had less HOOc radical scavenging activity than Trolox; nevertheless, at pH > 5.0, these acids reacted with the HOOc more quickly than the standard.According to the calculated data, in the pH range of 5.0-8.6, the CQAs had the highest HOOc antiradical activity (log(k overall ) = 5.1-6.2).It was found that the calculated rate constant for the 5CQA + HOOc reaction (k overall (calculation) = 3.10 × 10 5 M −1 s −1 ) exhibit a high level of consistence with the empirical observations (k exp = 1.28 × 10 5 M −1 s −1 , pH = 7.5). 17Therefore, the computed kinetic values are fairly accurate.

Conclusion
DFT calculations were performed to examine the effectiveness of monocaffeoylquinic acids in scavenging hydroperoxyl radicals.In water at physiological pH, the CQAs demonstrated z 10 4 times (k(water, pH = 7.4) = 1.27-5.32× 10 8 M −1 s −1 ) greater HOOc radical-trapping activity than in the nonpolar environment (k(lipid) = 1.09-1.93× 10 4 M −1 s −1 ).The FHT reaction of the neighboring hydroxyl group (O6 0 -H) of the dianion states determined the activity in the aqueous solution (G = 93.2-95.2%),while the SET mechanism of these states contributed 4.8-6.8% to the total rate constants.It is signicant that the computed rate constant of the HOOc radical-trapping activity in water at pH 7.5 agrees favorably with experimental ndings (k calculated /k experimental = 2.4), supporting the computational method.CQAs exhibited similar HOOc antiradical activities at pH < 3, however at higher pH values, 5CQA reaction with HOOc was slower than that of 3CQA or 4CQA.It was also found that CQAs had less HOOc radical scavenging activity than Trolox at pH 4.7 while at pH > 5.0 CQAs are better radical scavengers than the reference.The CQAs had the highest HOOc antiradical activity at pH = 5.0-8.6.Thus, in the physiological environments, the HOOc antiradical ability of CQAs is generally better than the reference antioxidants resveratrol, ascorbic acid, and Trolox.

Fig. 5
Fig. 5 AIM topological structures of the FHT TSs of the O6 0 -H bond of the anionic and dianionic states.The bond critical points (BCPs) are represented by red spheres, while the ring critical points (RCPs) are represented by yellow spheres.

Fig. 6
Fig. 6 Calculated log(k total ) (a) and log(k overall ) (b) at 298.15 K, in the CQAs + HOOc in water as a function of pH values.

Table 1
Calculated pK a and f a Ref. 66. b Calculated in this work.c f Molar fraction.